ABSTRACT There is a fundamental gap in knowledge of how the late stages of the hepatitis C virus (HCV) lifecycle (assembly, envelopment and secretion) are regulated. Existence of this gap represents an important problem as better understanding of these lifecycle stages could contribute important knowledge that could lead to new therapeutic strategies to target infection by enveloped viruses such as HCV. The long term goal of this proposal is to better understand the mechanisms that regulate the envelopment and secretion of HCV and more broadly, of RNA viruses. Several HCV proteins are known to associate in order to facilitate HCV envelopment. Our preliminary data suggest that the acidic domain of the small viral NS4A protein is also essential for envelopment. Further, we have found that NS4A interacts with an essential HCV secretion host factor, AP1M1, suggesting an additional role for NS4A in HCV secretion. Together, our data suggest unappreciated roles for the NS4A protein in both HCV envelopment and HCV secretion. Our central hypothesis is that NS4A modulates critical interactions with viral proteins to regulate HCV envelopment and with host proteins to facilitate HCV secretion. In order to define the function of NS4A in the late HCV lifecycle, we propose two specific aims. In aim 1, we will investigate the role of the NS4A acidic domain in coordinating interactions with viral proteins to regulate HCV envelopment. We will use an envelopment-defective NS4A to interrogate envelopment and determine how this mutation affects viral protein-protein interactions and localization. In aim 2, we will investigate NS4A interaction with the adaptor protein AP1M1. We will interrogate this interaction to determine the effect on NS4A and AP1M1 localization and determine how this interaction regulates trafficking of infectious virions through the secretory pathway. These studies will elucidate both viral protein-protein and viral-host protein interactions necessary for successful infection. This approach is innovative in that this will be the first study to define a mechanism for NS4A action in HCV envelopment and HCV secretion. The proposed research is significant because it will greatly contribute to our knowledge of HCV envelopment and HCV secretion, providing new insights into the mechanisms governing HCV infection. HCV is a member of the Flaviviridae family of viruses which also includes many established and emerging pathogens such as West Nile, Dengue and Zika Virus. Therefore, understanding HCV envelopment and secretion could also inform mechanisms of these processes in other RNA viruses. Ultimately, the proposed research has potential to inform envelopment and secretion studies across Flaviviridae research and provide strategies to combat these diseases.